A wheat bran polysaccharide, a preparation method thereof and application thereof in preparing frozen dough and frozen dough steamed buns

Abstract

The application provides a wheat bran polysaccharide and a preparation method and application thereof in preparing frozen dough and frozen dough steamed buns, and belongs to the technical field of food. The preparation method of the wheat bran polysaccharide comprises the following steps: mixing defatted wheat bran with water and cellulase, ultrasonic extraction, taking supernatant after enzyme inactivation, and drying to obtain the wheat bran polysaccharide. The preparation method of the wheat bran polysaccharide provided by the application adopts ultrasonic-enzyme synergistic extraction method, can make the extraction rate of the wheat bran polysaccharide reach 32.55%, the purity reach 80.80%, and make the prepared wheat bran polysaccharide be capable of improving the storage stability of the frozen dough and improving the quality of the frozen dough product, such as improving the hardness, elasticity, resilience and chewiness of the frozen dough steamed buns, improving the yellowness value of the frozen dough steamed buns and reducing the redness value.

Description

Technical Field

[0001] This invention belongs to the field of food technology, specifically relating to a wheat bran polysaccharide, its preparation method, and its application in the preparation of frozen dough and frozen dough steamed buns. Background Technology

[0002] Frozen dough technology not only effectively enables the large-scale and standardized production of fermented staple foods but also improves the transportation and storage efficiency of products such as steamed buns. However, freezing can cause degradation of the gluten protein network structure, leading to quality problems such as cracked dough surface and hard, dry core. Simultaneously, during frozen storage, the dough is affected by factors such as freezing rate and temperature fluctuations, causing ice crystals to increase in size and recrystallize, which damages the gluten protein network structure, impairs the starch surface structure, and reduces yeast activity, ultimately resulting in a decline in steamed bun quality. Wheat bran, due to its dense structure and hard texture, has a rough, bitter taste and is difficult to eat. Most wheat bran is used in brewing, vinegar making, and animal feed. Currently, the comprehensive utilization rate of wheat bran in the food industry is low, resulting in low economic value. Wheat bran has various health benefits; wheat bran polysaccharides are important bioactive substances in wheat bran, whose molecular chains are composed of different monosaccharide molecules linked by glycosidic bonds. Wheat bran polysaccharides contain monosaccharides such as glucose (Glu), arabinose (Ara), galactose (Gal), xylose (Xyl), mannose (Man), and rhamnose (Rha), and uronic acids such as galacturonic acid (Gal A) and glucuronic acid (Glu A). The monosaccharide composition and content of wheat bran polysaccharides are influenced by various factors, including wheat origin, species, pretreatment methods, and purification methods. Different extraction methods affect the monosaccharide composition of wheat bran polysaccharides, resulting in differences in the substitution of glycosidic bonds (single or double substitution) and the molecular weight of the polysaccharides. Consequently, different extraction methods lead to differences in their functional properties, structural composition, and solubility. The Hetao region is rich in wheat bran resources, but traditional polysaccharide extraction processes suffer from low efficiency and insufficient retention of active ingredients, severely restricting the high-value utilization of wheat bran resources. Currently, research on wheat bran polysaccharides in this field is relatively limited, and there are no reports on the correlation between wheat bran polysaccharides and the rheological properties, storage stability, and steamed bun quality characteristics of frozen dough. Summary of the Invention

[0003] In view of the technical problems existing in the prior art, such as the low comprehensive utilization rate of wheat bran in food, poor storage stability of frozen dough, and poor quality of frozen dough products, the present invention aims to provide a wheat bran polysaccharide that can improve the storage stability of frozen dough and the quality of frozen dough products.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for preparing wheat bran polysaccharide that can improve the storage stability of frozen dough and the quality of frozen dough products, comprising the following steps: mixing defatted wheat bran with water and cellulase, ultrasonically extracting, inactivating the enzyme, taking the supernatant, and drying to obtain wheat bran polysaccharide; the mass-volume ratio of defatted wheat bran to water is 1 g: 15 mL~35 mL; the addition ratio of defatted wheat bran to cellulase is 1 mg: 4500 U~5500 U.

[0005] Preferably, the ultrasonic extraction time is 10 min to 30 min, the ultrasonic extraction temperature is 40℃ to 60℃, and the ultrasonic intensity is 300 W to 600 W.

[0006] Preferably, the method for preparing defatted wheat bran includes the following steps: crushing and sieving wheat bran to obtain wheat bran powder; mixing and stirring the wheat bran powder with n-hexane, collecting the precipitate, and obtaining defatted wheat bran.

[0007] Preferably, the sieving is performed through a 50-70 mesh sieve; the mass-to-volume ratio of wheat bran powder to n-hexane is 1 g: 2 mL-5 mL; and the wheat bran is wheat bran from Yongliang No. 4 wheat in the Hetao region.

[0008] The present invention also provides a wheat bran polysaccharide that can improve the storage stability of frozen dough and the quality of frozen dough products, which is prepared by the above preparation method.

[0009] The present invention also provides the above-described preparation method or the application of the above-described wheat bran polysaccharide in the preparation of a frozen dough antifreeze agent.

[0010] The present invention also provides the above-described preparation method or the application of the above-described wheat bran polysaccharide in the preparation of frozen dough.

[0011] Preferably, the mass ratio of wheat bran polysaccharide to wheat flour is 0.5~1.5:100; the wheat bran polysaccharide can improve the storage stability of frozen dough; improving storage stability includes: reducing the freezeable water content of frozen dough, increasing water flowability, and improving the water-holding capacity of gluten protein; enhancing the viscoelasticity of frozen dough, improving the continuity and integrity of gluten protein network structure, and increasing the water absorption rate of frozen dough; prolonging the formation time and stabilization time of frozen dough; reducing the content of free sulfhydryl groups, increasing the content of disulfide bonds, and increasing the content of α-helix and β-sheet structures of gluten protein.

[0012] This invention also provides the above-described preparation method or the application of the above-described wheat bran polysaccharide in the preparation of frozen dough steamed buns.

[0013] Preferably, the mass ratio of wheat bran polysaccharide to wheat flour is 0.5~1.5:100; the wheat bran polysaccharide can improve the hardness, elasticity, resilience and chewiness of frozen dough steamed buns, and can increase the yellowness value of steamed buns and reduce the redness value of steamed buns.

[0014] The beneficial effects of this invention are: The method for preparing wheat bran polysaccharides provided by this invention employs an ultrasonic-enzyme synergistic extraction method, achieving an extraction rate of up to 32.55% and a purity of up to 80.80%. Furthermore, the prepared wheat bran polysaccharides can improve the storage stability of frozen dough and enhance the quality of frozen dough products, such as improving the hardness, elasticity, resilience, and chewiness of frozen dough steamed buns, and increasing the yellowness and decreasing the redness of frozen dough steamed buns. This invention provides a theoretical basis for the extraction of wheat bran polysaccharides and their application in frozen dough. Attached Figure Description

[0015] Figure 1 The effect of the material-to-liquid ratio on the extraction rate and purity of wheat bran polysaccharides; Figure 2 The effect of cellulase addition on the extraction rate and purity of wheat bran polysaccharides; Figure 3 The effect of extraction time on the extraction rate and purity of wheat bran polysaccharides; Figure 4 The effect of extraction temperature on the extraction rate and purity of wheat bran polysaccharides; Figure 5 The effect of ultrasonic intensity on the extraction rate and purity of wheat bran polysaccharides; Figure 6 The response surface plot (left) and contour plot (right) of polysaccharide extraction rate to the solid-liquid ratio and cellulase addition amount. Figure 7 The response surface plot (left) and contour plot (right) show the effect of material-liquid ratio and extraction time on polysaccharide extraction rate. Figure 8 Response surface plot (left) and contour plot (right) of cellulase addition amount and ultrasonic intensity on polysaccharide extraction rate; Figure 9 The response surface plot (left) and contour plot (right) of extraction time and ultrasonic intensity to polysaccharide extraction rate. Figure 10 The images show the chromatograms of a mixed monosaccharide standard and wheat bran polysaccharide ions, where A is the chromatogram of the mixed monosaccharide standard and B is the chromatogram of the monosaccharide components in wheat bran polysaccharide. Figure 11 The chromatogram of the molecular weight of wheat bran polysaccharides; Figure 12 Fourier transform infrared spectrum of wheat bran polysaccharide; Figure 13 The image shows the ultraviolet spectrum analysis of wheat bran polysaccharides. Figure 14 Polysaccharide 1 H nuclear magnetic resonance spectrum; Figure 15The effects of wheat bran polysaccharide on the rheological properties of frozen dough were investigated. Storage modulus (G') and loss modulus (G'') were calculated for A-10 d and B-10 d respectively, with wheat bran polysaccharide additions of 0.0%, 0.5%, 1.0%, 1.5%, and 2.0% for 10 days of frozen storage. Storage energy (G') and loss modulus (G'') were calculated for A-20 d and B-20 d respectively, with wheat bran polysaccharide additions of 0.0%, 0.5%, 1.0%, 1.5%, and 2.0% for 20 days of frozen storage. Storage energy (G') and loss modulus (G'') were calculated for A-30 d and B-30 d respectively, with wheat bran polysaccharide additions of 0.0%, 0.5%, 1.0%, 1.5%, and 2.0% for 30 days of frozen storage. Figure 16 The image shows the effect of wheat bran polysaccharide on the gluten network structure of frozen dough. FD refers to the fresh dough group, with freezing storage times of 10 d, 20 d, and 30 d; wheat bran polysaccharide addition amounts of 0.0%, 0.5%, 1.0%, 1.5%, and 2.0%; blue circles represent disruption of the gluten network structure; the images were taken using a laser confocal microscope at 400x magnification, with a scale bar of 100µm. Figure 17 The effect of wheat bran polysaccharides on the freezeable water content of frozen dough is shown, where different lowercase letters in the same column indicate significant differences between data. P <0.05); Figure 18 The effect of wheat bran polysaccharides on the moisture flowability of frozen dough; Figure 19 To determine the effect of wheat bran polysaccharides on the water-holding capacity of gluten protein, different lowercase letters indicate significant differences between data points. P <0.05); Figure 20 To determine the effect of wheat bran polysaccharides on the free sulfhydryl group content of gluten protein, different lowercase letters indicate significant differences between data. P <0.05); Figure 21 The effect of wheat bran polysaccharides on the color of frozen dough steamed buns was investigated, with wheat bran polysaccharide addition amounts of 0.0%, 0.5%, 1.0%, 1.5%, and 2.0%; A represents brightness L. As a result, B is red-green a As a result, C is yellow-blue. result; Figure 22 The effect of wheat bran polysaccharide on the textural properties of frozen dough steamed buns was investigated. The wheat bran polysaccharide addition amounts were 0.0%, 0.5%, 1.0%, 1.5%, and 2.0%, respectively. Among them, A represents hardness, B represents elasticity, C represents chewiness, and D represents resilience. Figure 23The effect of wheat bran polysaccharide addition on the sensory evaluation of frozen steamed buns was investigated, where FSB refers to fresh steamed buns, and 0.0%, 0.5%, 1.0%, 1.5%, and 2.0% refer to the amount of wheat bran polysaccharide added. Figure 24 The study investigated the effects of wheat bran polysaccharides (FSB) on the microstructure of frozen dough steamed buns. FSB refers to fresh steamed buns. Freezing times were 10 days, 20 days, and 30 days. The amounts of wheat bran polysaccharides added were 0.0%, 0.5%, 1.0%, 1.5%, and 2.0%. Figure 25 XRD patterns of frozen dough steamed buns with different wheat bran polysaccharide additions are shown. FSB represents fresh steamed buns; 10 d, 20 d, and 30 d represent frozen storage time, respectively; and the wheat bran polysaccharide additions are 0.0%, 0.5%, 1.0%, 1.5%, and 2.0%, respectively. Detailed Implementation

[0016] This invention provides a method for preparing wheat bran polysaccharide that can improve the storage stability of frozen dough and the quality of frozen dough products, comprising the following steps: mixing defatted wheat bran with water and cellulase, ultrasonically extracting, inactivating the enzyme, taking the supernatant, and drying to obtain wheat bran polysaccharide; the mass-volume ratio of defatted wheat bran to water is 1 g: 15 mL~35 mL; the addition ratio of defatted wheat bran to cellulase is 1 mg: 4500 U~5500 U.

[0017] In this invention, the preferred method for preparing defatted wheat bran includes the following steps: pulverizing and sieving the wheat bran to obtain wheat bran powder; mixing and stirring the wheat bran powder with n-hexane, collecting the precipitate, and obtaining defatted wheat bran. The sieving is preferably done through a 50-70 mesh sieve, more preferably through a 60 mesh sieve; the mass-to-volume ratio of the wheat bran powder to n-hexane is preferably 1 g: 2 mL-5 mL, more preferably 1 g: 3 mL-4 mL; the wheat bran is preferably from Yongliang No. 4 wheat in the Hetao region. In this invention, when using n-hexane for defatting, it is preferably performed twice: mixing the wheat bran powder with n-hexane, stirring with a magnetic stirrer, centrifuging, removing the supernatant, adding n-hexane again at the same material-to-liquid ratio, stirring, centrifuging, collecting the precipitate, and drying to obtain defatted wheat bran.

[0018] In this invention, the preferred mass-to-volume ratio of defatted wheat bran to water is 1 g: 20 mL to 30 mL, and the preferred ratio of defatted wheat bran to cellulase is 1 mg: 4800 U to 5200 U. This invention does not specifically limit the source of the cellulase; commercially available products commonly used in the field are acceptable. In this invention, the preferred ultrasonic extraction time is 10 min to 30 min, more preferably 15 min to 25 min; the preferred ultrasonic extraction temperature is 40℃ to 60℃, more preferably 45℃ to 55℃; and the preferred ultrasonic intensity is 300 W to 600 W, more preferably 400 W to 500 W. In this invention, the preferred method of enzyme inactivation is boiling water bath inactivation, and the preferred inactivation time is 10 min. In this invention, centrifugation is required after enzyme inactivation, and the preferred centrifugation conditions are 4000 r / min for 15 min.

[0019] This invention also provides a wheat bran polysaccharide that can improve the storage stability of frozen dough and the quality of frozen dough products, prepared by the above-described preparation method. Ion chromatography analysis of the wheat bran polysaccharide prepared according to the method of this invention shows that the monosaccharide composition is mainly glucose (84.00%), followed by xylose (7.13%), arabinose (4.86%), and galactose (4.01%). Gel permeation chromatography analysis shows that the number-average molecular weight (Mn) is 128.4 kDa, the weight-average molecular weight (Mw) is 235.0 kDa, and the polydispersity index (Mw / Mn) is 1.83. Fourier transform infrared spectroscopy and nuclear magnetic resonance results indicate that the wheat bran polysaccharide comprises monosaccharide components of glucose, xylose, arabinose, and galactose.

[0020] This invention also provides the above-described preparation method or the application of the above-described wheat bran polysaccharide in the preparation of a frozen dough antifreeze agent. This invention is the first to propose that the wheat bran polysaccharide prepared by the method of this invention can be used as a novel frozen dough antifreeze agent.

[0021] The present invention also provides the above preparation method or the application of the above wheat bran polysaccharide in the preparation of frozen dough, wherein the mass ratio of wheat bran polysaccharide to wheat flour is preferably 0.5~1.5:100, more preferably 0.9~1.1:100, and even more preferably 1:100.

[0022] The wheat bran polysaccharide provided by this invention can improve the storage stability of frozen dough. Compared with the control group without additives, the addition of wheat bran polysaccharide reduces the freezeable water content of frozen dough, increases water flowability, and significantly improves the water-holding capacity of gluten protein (P<0.05). At the same time, the viscoelasticity of the dough is enhanced, the continuity and integrity of the gluten protein network structure are improved, and the water absorption rate of the dough is increased. When the addition amount is 1.0%, the dough formation time is extended from 2.76 min to 3.16 min, and the stability time is extended from 6.40 min to 7.25 min. The content of free thiol groups is reduced, the content of disulfide bonds is increased, and the content of α-helix and β-sheet structures of gluten protein increases.

[0023] The present invention also provides the above preparation method or the application of the above wheat bran polysaccharide in the preparation of frozen dough steamed buns, wherein the mass ratio of wheat bran polysaccharide to wheat flour is preferably 0.5~1.5:100, more preferably 0.9~1.1:100, and even more preferably 1:100.

[0024] In this invention, the addition of wheat bran polysaccharide can improve the hardness, elasticity, resilience, and chewiness of frozen dough steamed buns, increasing the yellowness and decreasing the redness of the buns. When the addition amount is 1.0%, the steamed buns have the highest sensory score. The specific volume of the steamed buns stored frozen for 10, 20, and 30 days increased from 2.50 mL / g to 2.72 mL / g, 2.26 mL / g to 2.56 mL / g, and 1.65 mL / g to 2.39 mL / g, respectively. The aspect ratio increased from 0.67% to 0.71%, 0.65% to 0.70%, and 0.59% to 0.63%, respectively. Scanning electron microscopy and X-ray diffraction analysis showed that wheat bran polysaccharide can promote the densification of the gluten network, inhibit starch molecular chain interactions, reduce crystallinity, and effectively alleviate freezing damage.

[0025] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0026] Unless otherwise specified, the following embodiments are all conventional methods.

[0027] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0028] The wheat bran used in the following examples was from Yongliang No. 4 wheat from the Hetao region, provided by the Bayannur Institute of Agricultural and Animal Husbandry Sciences, Inner Mongolia. Cellulase was purchased from Shanghai Yuanye Biotechnology Co., Ltd. Yeast was purchased from Angel Yeast Co., Ltd.

[0029] The data processing for the following examples and experimental cases was as follows: each experiment consisted of three replicates, and all experimental data were expressed as mean ± standard deviation. Excel 2016 was used for statistical analysis, and SPSS 22.0 was used for significance analysis and analysis of variance. P <0.05 is considered a significant difference. P <0.01 indicates a highly significant difference.

[0030] Example 1 A wheat bran polysaccharide that can improve the storage stability of frozen dough and the quality of frozen dough products is prepared by the following method: Wheat bran was passed through a 60-mesh sieve to remove residual wheat flour. The wheat bran was then pulverized using a cup mill and passed through a 60-mesh sieve again to obtain wheat bran powder. The wheat bran powder was mixed with n-hexane at a material-to-liquid ratio of 1 g: 3 mL. After stirring with a magnetic stirrer, the mixture was centrifuged at 3000 r / min for 10 min. The supernatant was removed. Then, n-hexane was added again at a material-to-liquid ratio of 1 g: 3 mL (wheat bran powder: n-hexane). After stirring, the mixture was centrifuged at 3000 r / min for 10 min. The precipitate was collected and dried in a fume hood to obtain defatted wheat bran.

[0031] Weigh defatted wheat bran and place it in a centrifuge tube. Add distilled water at a material-to-liquid ratio of 1 g: 28 mL. Add cellulase at a ratio of 1 mg: 4874 U of defatted wheat bran to cellulase. Extract the mixture using an ultrasonic processor at 526 W and 50 °C for 21 min. Then, inactivate the extract in a boiling water bath at 100 °C for 10 min. Centrifuge at 4000 r / min for 15 min and collect the supernatant. Freeze-dry the supernatant to obtain wheat bran polysaccharide.

[0032] Example 2 A wheat bran polysaccharide that can improve the storage stability of frozen dough and the quality of frozen dough products is prepared by the following method: Wheat bran was passed through a 50-mesh sieve to remove residual wheat flour. The wheat bran was then pulverized using a cup mill and passed through a 50-mesh sieve to obtain wheat bran powder. The wheat bran powder was mixed with n-hexane at a material-to-liquid ratio of 1 g: 2 mL. After stirring with a magnetic stirrer, the mixture was centrifuged at 3000 r / min for 10 min. The supernatant was removed. Then, n-hexane was added again at a material-to-liquid ratio of 1 g: 2 mL (wheat bran powder: n-hexane). After stirring, the mixture was centrifuged at 3000 r / min for 10 min. The precipitate was collected and dried in a fume hood to obtain defatted wheat bran.

[0033] Weigh defatted wheat bran and place it in a centrifuge tube. Add distilled water at a material-to-liquid ratio of 1 g:15 mL. Add cellulase at a ratio of 1 mg:4500 U of defatted wheat bran to cellulase. Extract the mixture using an ultrasonic processor at 300 W and 40 °C for 30 min. Then, inactivate the extract in a boiling water bath at 100 °C for 10 min. Centrifuge at 4000 r / min for 15 min and collect the supernatant. Freeze-dry the supernatant to obtain wheat bran polysaccharide.

[0034] Example 3 A wheat bran polysaccharide that can improve the storage stability of frozen dough and the quality of frozen dough products is prepared by the following method: Wheat bran was passed through a 70-mesh sieve to remove residual wheat flour. The wheat bran was then pulverized using a cup mill and passed through a 70-mesh sieve to obtain wheat bran powder. The wheat bran powder was mixed with n-hexane at a material-to-liquid ratio of 1 g: 5 mL. After stirring with a magnetic stirrer, the mixture was centrifuged at 3000 r / min for 10 min. The supernatant was removed. Then, n-hexane was added again at a material-to-liquid ratio of 1 g: 5 mL (wheat bran powder: n-hexane). After stirring, the mixture was centrifuged at 3000 r / min for 10 min. The precipitate was collected and dried in a fume hood to obtain defatted wheat bran.

[0035] Weigh defatted wheat bran and place it in a centrifuge tube. Add distilled water at a material-to-liquid ratio of 1 g:35 mL. Add cellulase at a ratio of 1 mg:5500 U of defatted wheat bran to cellulase. Extract the mixture using an ultrasonic processor at 600 W and 60 °C for 10 min. Then, inactivate the extract in a boiling water bath at 100 °C for 10 min. Centrifuge at 4000 r / min for 15 min and collect the supernatant. Freeze-dry the supernatant to obtain wheat bran polysaccharide.

[0036] Experimental Example 1 Single-factor experimental design for the preparation of wheat bran polysaccharides (1) Effect of material-liquid ratio on polysaccharide extraction rate Under the conditions of 4000 U / mg cellulase addition, 30 min extraction time, 50℃ extraction temperature, and 200 W ultrasonic intensity, experiments were conducted with material-to-liquid ratios of 1:15, 1:25, 1:35, 1:45, and 1:55 g / mL, respectively. All other conditions were the same as in Example 1. The effects of different material-to-liquid ratios on polysaccharide extraction rate were investigated, with wheat bran polysaccharide purity and extraction rate as the standards.

[0037] Determination of purity and extraction rate of wheat bran polysaccharides: The purity of wheat bran polysaccharides was determined by the phenol-sulfuric acid method.

[0038] Purity of wheat bran polysaccharides (%) 100 Where: m1 is the sugar content of the sample, μg; m2 is the sample mass, g; V1 is the sample volume at which it is brought to a final volume, mL; V2 is the sample volume taken during the determination, mL; 0.9 is the correction factor for converting glucose to dextran.

[0039] Extraction rate of wheat bran polysaccharides (%) 00 In the formula: M1 is the mass of wheat bran polysaccharide, g; M2 is the mass of defatted wheat bran, g; X is the purity of wheat bran polysaccharide, %.

[0040] The results are as follows Figure 1 As shown, the extraction rate and purity of wheat bran polysaccharides initially increased and then decreased with increasing material-to-liquid ratio. The extraction rate and purity reached their peak values ​​of 28.05% and 71.09%, respectively, when the material-to-liquid ratio was 1:25 g / mL. This initial increase followed by a decrease in extraction rate and purity may be because the amount of distilled water added affects the concentration gradient and intermolecular interactions of the wheat bran polysaccharide solution. Insufficient distilled water results in a higher substrate concentration and a smaller concentration gradient, weakening the intermolecular interaction effect. Conversely, excessive distilled water leads to a lower substrate concentration and a larger concentration gradient, thus affecting the dissolution of wheat bran polysaccharides. Therefore, a material-to-liquid ratio of 1:25 g / mL was chosen.

[0041] (2) Effect of cellulase addition on polysaccharide extraction rate Under the conditions of a material-to-liquid ratio of 1:35 g / mL, an extraction time of 30 min, an extraction temperature of 50℃, and an ultrasonic intensity of 200 W, cellulase addition amounts of 3500, 4000, 4500, 5000, and 5500 U / mg were selected for the experiment, with the rest being the same as above (1), to explore the effect of different cellulase addition amounts on polysaccharide extraction rate.

[0042] The results are as follows Figure 2 As shown, with the increase of cellulase addition, the extraction rate and purity of wheat bran polysaccharides showed a trend of first increasing and then decreasing. When the cellulase addition was 5000 U / mg, the extraction rate and purity of wheat bran polysaccharides reached their maximum values, at 29.49% and 68.09%, respectively. When the cellulase addition was higher than 5000 U / mg, the extraction rate and purity of wheat bran polysaccharides decreased because the cellulase concentration had reached saturation, and almost all of the wheat bran polysaccharides dissolved, leaving the remaining cellulase unable to function. Therefore, a cellulase addition of 5000 U / mg was chosen.

[0043] (3) Effect of extraction time on polysaccharide extraction rate Under the conditions of a material-to-liquid ratio of 1:35 g / mL, a cellulase addition of 4000 U / mg, an extraction temperature of 50℃, and an ultrasonic intensity of 200 W, extraction times of 10, 20, 30, 40, and 50 min were selected for the experiment, with the rest being the same as above (1), to explore the effect of different times on the polysaccharide extraction rate.

[0044] The results are as follows Figure 3 As shown, the extraction rate and purity of wheat bran polysaccharides first increased and then decreased with increasing extraction time. The extraction rate and purity reached their maximum values ​​of 25.11% and 66.97%, respectively, when the extraction time was 20 min. At a certain ultrasonic frequency, an appropriate ultrasonic time promotes the dissolution of polysaccharides, possibly because ultrasonic vibration accelerates the entry of polysaccharides into the solvent. However, excessively long ultrasonic times can damage the polysaccharide structure, leading to the accelerated release of polar substances from cells, while non-polar substances and other impurities also dissolve simultaneously. This rapidly reduces the concentration difference between the solute and the solution, weakening the driving force for further solute dissolution and thus decreasing the extraction rate and purity of wheat bran polysaccharides. Therefore, an extraction time of 20 min was chosen.

[0045] (4) Effect of extraction temperature on polysaccharide extraction rate Under the conditions of a material-to-liquid ratio of 1:35 g / mL, a cellulase addition of 4000 U / mg, an extraction time of 30 min, and an ultrasonic intensity of 200 W, the extraction temperatures of 30, 40, 50, 60 and 70℃ were selected for the experiment, and the rest were the same as above (1) to explore the effect of different temperatures on the polysaccharide extraction rate.

[0046] The results are as follows Figure 4 As shown, the extraction rate and purity of wheat bran polysaccharides first increased and then decreased with increasing extraction temperature. The extraction rate and purity reached their maximum values ​​of 20.31% and 63.70%, respectively, at an extraction temperature of 50℃. Within a certain temperature range, the increase in extraction rate and purity of wheat bran polysaccharides with increasing temperature may be due to enhanced cellulase activity; it may also be due to increased molecular thermal motion and mass transfer efficiency, leading to a faster polysaccharide dissolution rate. When the extraction temperature exceeded 50℃, the extraction rate and purity of wheat bran polysaccharides decreased, possibly due to the high temperature damaging the polysaccharide structure or inhibiting cellulase activity. Therefore, an extraction temperature of 50℃ was selected.

[0047] (5) Effect of ultrasonic intensity on polysaccharide extraction rate Under the conditions of a material-to-liquid ratio of 1:35 g / mL, a cellulase addition of 4000 U / mg, an extraction time of 30 min, and an extraction temperature of 50℃, ultrasonic intensities of 200, 300, 400, 500, and 600 W were selected for the experiment, with the rest being the same as above (1), to explore the effect of different ultrasonic intensities on the polysaccharide extraction rate.

[0048] The results are as follows Figure 5 As shown, the extraction rate and purity of wheat bran polysaccharides first increased and then decreased with increasing ultrasonic intensity. When the ultrasonic intensity was 500 W, the extraction rate and purity of wheat bran polysaccharides reached their maximum values ​​of 33.06% and 76.07%, respectively. The increase in the extraction rate and purity of wheat bran polysaccharides may be because the cavitation effect induced by ultrasonic vibration effectively disrupts the cell wall structure of wheat bran, thereby promoting the release of polysaccharide components and accelerating the diffusion process between molecules. This not only increases the contact area between the substrate and cellulase but also effectively improves the extraction rate of wheat bran polysaccharides. When the ultrasonic power exceeds a certain limit, it will damage the polysaccharide structure, leading to a decrease in the extraction rate and purity of wheat bran polysaccharides. Therefore, an ultrasonic intensity of 500 W was selected.

[0049] Experimental Example 2 Response surface methodology Based on the optimization results of the single-factor experimental parameters in Experiment 1, a Box-Behnken Design (BBD) response surface model was constructed using Design-Expert 12 software. The key process parameters were set as follows: material-to-liquid ratio 1:25 g / mL, cellulase addition 5000 U / mg, extraction time 20 min, and ultrasonic intensity 500 W. The experiment was designed using a four-factor, three-level analysis method, as shown in Table 1.

[0050] Table 1. Response Surface Experiment Factors and Level Design

[0051] (1) Establishment of response surface model and analysis of variance A mathematical model was constructed based on response surface methodology. Combining the results of single-factor experiments, the material-to-liquid ratio (A), cellulase dosage (B), extraction time (C), and ultrasonic intensity (D) were set as independent variables, and the wheat bran polysaccharide yield (Y) was used as the response index. A Box-Behnken experimental design was employed, and regression analysis was performed on the experimental data using Design-Expert 12 to obtain second-order polynomial equations describing the relationship between each factor and the response value. Y=+31.56+0.4758A-0.5167B+0.3792C+0.5100D-1.08AB+1.74AC+0.5300AD+0.1600BC+0.6250BD-0.3850CD-1.56A 2-1.82B 2 -1.81C 2 -1.54D 2 .

[0052] The results are shown in Table 2, and the significance test and variance analysis results of the regression equation are shown in Table 3.

[0053] Table 2. Results and Design of Response Surface Experiment

[0054] Table 3. Results of Analysis of Variance

[0055] Note: The difference is extremely significant ( P <0.01); Significant difference ( P <0.05).

[0056] As can be seen from Table 3, P A value less than 0.01 indicates that the model is highly significant and fits reality well. R0 2 A value of 0.9504 indicates that 95.04% of the change in the model's response value comes from the change in the selected dependent variable, demonstrating a high correlation between the response value extraction rate and the actual value. Meanwhile, the model's lack-of-fit term... P The value is 0.4875, which is greater than 0.05, indicating that the model is not poorly fitted and can therefore be used to predict the actual extraction rate of the response value. The order of influence of each factor on the extraction rate of wheat bran polysaccharides is: ultrasonic intensity > cellulase addition > material-liquid ratio > extraction time. Among them, factors A, B, D, AB, AC, BD, CD, and A... 2 B 2 C 2 D 2 The effect on the response value is extremely significant. P The value is less than 0.01.

[0057] (2) Response surface analysis Design-Expert 12 software was used to locate the response surface plot, and the interactions between variables were interpreted using a 3D response surface and a 2D contour plot. The greater the influence, the steeper the surface. The results are as follows: Figures 6-9 As shown.

[0058] Depend on Figure 6It can be seen that when the extraction time and ultrasonic intensity are fixed, the slope of the three-dimensional response surface plot between the solid-liquid ratio (A) and the amount of cellulase added (B) is relatively large, and the contour plot shows an elliptical diffusion. With increasing solid-liquid ratio and cellulase addition, the polysaccharide content in the extract gradually increases. The polysaccharide content reaches its maximum when the solid-liquid ratio is 1:25 g / mL and the cellulase addition is 5000 U / mg. Subsequent increases in the solid-liquid ratio and cellulase addition lead to a decrease in polysaccharide content, showing a trend of first increasing and then decreasing. The results indicate a certain interaction between the two factors.

[0059] Depend on Figure 7 It can be seen that when the amount of cellulase added and the ultrasonic intensity are fixed, the slope of the three-dimensional response surface plot between the solid-liquid ratio (A) and the extraction time (C) is relatively large, and the contour plot shows an elliptical diffusion. With the gradual increase of the solid-liquid ratio and extraction time, the polysaccharide content in the extract gradually increases, reaching a peak when the solid-liquid ratio reaches 1:25 g / mL and the extraction time reaches 20 min. Thereafter, further increases in the solid-liquid ratio and extraction time lead to a decrease in polysaccharide content, showing an overall trend of first increasing and then decreasing. The experimental results indicate that there is an interaction between the solid-liquid ratio and extraction time, both factors jointly affecting the polysaccharide extraction rate.

[0060] Depend on Figure 8 It can be seen that when the material-to-liquid ratio and extraction time are fixed, the slope of the three-dimensional response surface plot between the cellulase addition amount (B) and the ultrasonic intensity (D) is relatively large, and the contour plot shows an elliptical diffusion. Gradually increasing the cellulase addition amount and ultrasonic intensity, the polysaccharide content in the extract begins to increase. When the cellulase addition amount is 5000 U / mg and the ultrasonic intensity reaches 500 W, the polysaccharide content in the extract reaches its peak. Further increasing the cellulase addition amount and ultrasonic intensity causes the polysaccharide content to decrease, showing an overall trend of first increasing and then decreasing. The experimental results show that there is a certain interaction effect between the cellulase addition amount and ultrasonic intensity; both factors work synergistically in the polysaccharide extraction process and jointly determine the polysaccharide extraction rate.

[0061] Depend on Figure 9 It can be seen that when the amount of cellulase added and the material-to-liquid ratio are fixed, the slope of the three-dimensional response surface plot between extraction time (C) and ultrasonic intensity (D) is relatively large, and the contour plot shows an elliptical diffusion. Gradually increasing the extraction time and ultrasonic intensity leads to a gradual increase in the polysaccharide content in the extract. When the extraction time is extended to 20 min and the ultrasonic intensity is increased to 500 W, the polysaccharide content reaches its peak, indicating that these two factors have a synergistic optimization effect in promoting polysaccharide dissolution. However, beyond this threshold, the polysaccharide content begins to decrease, showing a trend of first increasing and then decreasing. The experimental results show that there is a certain interaction between extraction time and ultrasonic intensity; they are interrelated and mutually influential.

[0062] (3) Optimal condition prediction and verification experiment Using Design-Expert 12 software, the optimal conditions for extracting wheat bran polysaccharides were determined: a material-to-liquid ratio of 1:27.539 g / mL, a cellulase dosage of 4873.887 U / mg, an extraction time of 20.795 min, and an ultrasonic intensity of 525.989 W. Under these conditions, the theoretical extraction rate of wheat bran polysaccharides was 28.63%. Considering the feasibility of practical operation, the above conditions were adjusted to a material-to-liquid ratio of 1:28 g / mL, a cellulase dosage of 4874 U / mg, an extraction time of 21 min, and an ultrasonic intensity of 526 W for verification experiments. Three parallel experiments were set up, and the actual measured extraction rate of wheat bran polysaccharides was 32.55%, which was close to the predicted value, indicating that the theoretical prediction value of the model is consistent with the actual value.

[0063] Experimental Example 3 Determination of protein and dietary fiber content of wheat bran polysaccharides obtained in Example 1 Protein determination: GB 5009.5-2016 (Method I). Dietary fiber determination: GB 5009.88-2023 (Enzymatic gravity method).

[0064] The results are shown in Table 4. The content of wheat bran polysaccharide was 80.80%, total dietary fiber (TDF) was 10.83%, soluble dietary fiber (SDF) was 6.39%, and insoluble dietary fiber (IDF) was 4.11%. This indicates that wheat bran polysaccharide is rich in dietary fiber, with soluble dietary fiber accounting for a large proportion of the total dietary fiber. Dietary fiber has high water-holding and water-absorbing properties, which can enable dough to form a viscous gel network structure, thereby improving the uniformity and elasticity of the dough, retaining more moisture, inhibiting excessive starch swelling and diffusion, increasing the water content of the dough, and reducing its quality deterioration during storage. Meanwhile, wheat bran polysaccharide contains 10.80% protein, possibly because the polysaccharide has not undergone purification treatment.

[0065] Table 4. Composition and content of wheat bran polysaccharides

[0066] Test Example 4 Characterization results of wheat bran polysaccharide obtained in Example 1 (1) Determination of monosaccharide composition Accurately weigh the wheat bran polysaccharide sample obtained in Example 1, and acid-hydrolyze it with 2 mol / L trifluoroacetic acid (TFA) (121℃, 2 h), then blow it dry with nitrogen. Wash with methanol (repeated 3 times) to remove residual acid, reconstitute with ultrapure water, and then perform chromatographic analysis. Monosaccharide components were determined using a Thermo ICS 5000+ ion chromatography system (electrochemical detector), and filtered through a 0.22 μm filter membrane before injection.

[0067] Chromatographic analysis was performed using a Dionex™ CarboPac™ PA20 column (150 × 3 mm, 10 μm) with an injection volume of 5 μL. Mobile phase A was water, mobile phase B was 0.1 mol / L sodium hydroxide (NaOH), and mobile phase C was solution B with added 0.2 mol / L sodium acetate (NaAc). The flow rate was 0.5 mL / min, and the column temperature was 30 °C with gradient elution. Elution gradient: 0 min A / B / C phase (95:5:0, V / V), 26 min A / B / C phase (85:5:10, V / V), 42 min A / B / C phase (85:5:10, V / V), 42.1 min A / B / C phase (60:0:40, V / V), 52 min A / B / C phase (60:40:0, V / V), 52.1 min A / B / C phase (95:5:0, V / V), 60 min A / B / C phase (95:5:0, V / V).

[0068] The results are as follows Figure 10 As shown in Table 5, wheat bran polysaccharide is a polysaccharide composed of arabinose, galactose, glucose, and xylose in different molar percentages, with retention times of 9.94 min, 12.79 min, 14.66 min, and 17.54 min, respectively. Table 5 shows that glucose is the main monosaccharide in wheat bran polysaccharide, accounting for 84% of the total molar percentage. In addition, there are certain amounts of arabinose, galactose, and xylose. The main component of wheat bran polysaccharide in this invention is glucose, which differs significantly from existing publicly available research results on wheat bran polysaccharides in this field. In summary, due to differences in wheat varieties, extraction methods, and other factors, the content of each monosaccharide can vary.

[0069] Table 5 Monosaccharide composition of wheat bran polysaccharides

[0070] (2) Molecular weight determination The determination was performed using gel permeation chromatography (under heating and dissolution conditions). Chromatographic column: PL aquagel-OH (7.5 × 50 mm, 8 μm), PL aquagel-OH (7.5 × 300 mm, 8 μm) in series. Detector: Differential refractive index detector. Conditions and reagents: Test temperature 45℃, solvent flow rate 1 mL / min, solvent 0.1 mol / mL NaNO3, standard: polyethylene glycol (PEG), injection volume 20 μL.

[0071] The results are as follows Figure 11As shown in Table 6, the peak molecular weight represents the most abundant molecular group in the sample, reflecting the concentrated distribution trend of molecular weight. Number-average molecular weight is the calculated average of the molecular weights of all molecules in the polysaccharide population, reflecting the average characteristics of molecular quantity and suitable for polysaccharide systems with narrow molecular weight distributions. Weight-average molecular weight often characterizes polysaccharide samples with broad molecular weight distributions. Z-average molecular weight has extremely strong selectivity for high molecular weight molecules and is often used to characterize the breadth of polysaccharide molecular weight distribution. Polydispersity index is a core parameter characterizing the breadth of polysaccharide molecular weight distribution, reflecting the uniformity of polysaccharide molecular weight distribution; the lower the polydispersity index, the better the homogeneity of the polysaccharide. Table 6 shows that the number-average molecular weight (Mn) of wheat bran polysaccharide is 128.4 kDa, the weight-average molecular weight (Mw) is 235.0 kDa, and the polydispersity index is 1.83. Compared with the molecular weights of plant polysaccharides reported in other studies, the molecular weight of wheat bran polysaccharide in this invention is relatively low, possibly because the cavitation effect of ultrasound reduces the molecular weight of the polysaccharide.

[0072] Table 6. Molecular weight distribution of wheat bran polysaccharides

[0073] (3) Fourier transform infrared spectroscopy determination Sample preparation was performed using the potassium bromide tableting method: the sample was ground and mixed with dry potassium bromide powder, then a transparent thin film was prepared using a tablet press. Fourier transform infrared spectroscopy was used for detection, with parameters set to a resolution of 4 cm⁻¹. -1 32 scans, wavenumber range 400~4000 cm⁻¹ -1 .

[0074] The types of monosaccharides, glycosidic bonds, and functional groups in the polysaccharide structure can be evaluated using Fourier transform infrared spectroscopy. Results are as follows: Figure 12 As shown, the infrared spectrum of wheat bran polysaccharides is displayed in the range of 4000~400 cm⁻¹. -1 The characteristic absorption peak is at 3420 cm⁻¹. -1 Near 2900 cm⁻¹, the absorption peak is caused by the OH vibration; at 2900 cm⁻¹ -1 The absorption peak near the 1600 cm⁻¹ is caused by the stretching vibration of CH; -1 The absorption peak near the 1400 cm⁻¹ is due to the carbonyl (C=O) stretching vibration; -1 Nearby, there is a carbon-hydrogen bond bending vibration. The above four peaks are consistent with the characteristic infrared absorption peaks of polysaccharides. 1000~1200 cm⁻¹ -1 The three nearby absorption bands indicate the presence of pyran and furan glycosidic bonds. Furthermore, at 1100 cm⁻¹... -1 Most of the following absorption peaks are likely related to monosaccharides, at 1040 cm⁻¹. -1 The nearby peaks are characteristic of arabinose (Ara), located at 950–800 cm⁻¹.-1 At that time, absorption peaks of glucose (Glc) and galactose (Gal) were observed. The results are consistent with the monosaccharide composition analysis in the results of this invention (Table 5).

[0075] (4) Ultraviolet spectroscopy measurement Prepare a 20 μg / mL sample solution of wheat bran polysaccharide, and use distilled water as a blank control to calibrate the scanning baseline. Perform a full-wavelength scan (200–800 nm) using a UV spectrophotometer to detect characteristic absorption peaks.

[0076] The UV-Vis spectrophotometer can effectively identify whether polysaccharide samples contain biomolecules such as proteins and nucleic acids. 260 nm and 280 nm are the maximum UV absorption wavelengths for nucleic acids and proteins, respectively. The results are as follows: Figure 13 As shown, absorption peaks appear in the spectra at 260 nm and 280 nm, indicating the presence of a small amount of protein in the crude polysaccharide, consistent with the previous results of this experiment (Table 4). This may be due to the absorption of ultraviolet light by the conjugated double bonds of tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe) residues in the protein molecule.

[0077] (5) Nuclear magnetic resonance analysis Nuclear magnetic resonance (NMR) samples were prepared using a deuterated solvent displacement method: 30 mg of sample was accurately weighed, subjected to three deuterated solvent displacement treatments, and then dissolved in 0.5 mL of heavy water (D₂O). (H₂O spectrum) 1 The H NMR detection was performed under a constant temperature of 25℃, and the one-dimensional proton signal of the sample solution was directly acquired using a 600 MHz nuclear magnetic resonance spectrometer.

[0078] Nuclear magnetic resonance (NMR) was used to detect the NMR signals of hydrogen nuclei in polysaccharide molecules to analyze the anomeric configuration, glycosidic bond type, and linkage sequence of the polysaccharides. This experiment employed... 1 1H NMR analysis of wheat bran polysaccharides 1 In H NMR spectra, the proton signal of the anomeric carbon of polysaccharides is generally in the range of δ 4.80~5.5 ppm, as shown in the following results. Figure 14 As shown, the multiple peaks within the interval mainly correspond to the protons on the sugar ring in the wheat bran polysaccharide molecule, and their chemical shifts are influenced by the combined effects of the glycosidic bond type, sugar ring configuration, and adjacent sugar groups. α Type and β The chemical shifts of the terminal isomer hydrogens of the glycosidic bond generally range from 4.4 to 5.8 ppm. The signals at 5.13 ppm and 5.31 ppm for wheat bran polysaccharide are respectively... α -D-Gal and α The characteristic peak of the -L-Gal anomeric hydrogen is observed in the range of 4.97 ppm to 5.44 ppm. 1HNMR signal represents α -D-GalAp and α -L-Araf residues; 1,4- were observed at 4.70 ppm, 4.56 ppm and 4.12 ppm, respectively. β -D-Galp、1,3- β -D-Galp and 1,3,6- β -D-Galp is a typical signal. Infrared spectroscopy, monosaccharide composition, and nuclear magnetic resonance results suggest that wheat bran polysaccharide is composed of →4)- β -D-Glcp-(1→connecting unit is the main one).

[0079] Example 4 A frozen dough, prepared as follows: Based on 100 g of wheat flour, dissolve 0.7 g (0.7%) of yeast in warm water, mix 1.3 g of wheat bran polysaccharide obtained in Example 1 with wheat flour and place it in a dough mixer, add the activated yeast and 57 g (57%) of warm water at 30°C, knead the dough with the dough mixer, press it repeatedly 10 times on a sheet press, shape it and place it in a freezer box to freeze at -40°C for 2 hours, and then transfer it to -18°C for frozen storage.

[0080] Example 5 A frozen dough, prepared as follows: Based on 100 g of wheat flour, dissolve 0.7 g (0.7%) of yeast in warm water, mix 1.2 g of wheat bran polysaccharide obtained in Example 1 with wheat flour and place it in a dough mixer, add the activated yeast and 57 g (57%) of warm water at 30°C, knead the dough with the dough mixer, press it repeatedly 10 times on a sheet press, shape it and place it in a freezer box to freeze at -40°C for 2 hours, and then transfer it to -18°C for frozen storage.

[0081] Experimental Example 5 The difference from Example 4 is that the amount of wheat bran polysaccharide added in Example 1 was 0.0g, 0.5g, 1.0g, 1.5g, and 2.0g respectively (calculated based on the amount of wheat flour added, the proportions of wheat bran polysaccharide added were 0.0%, 0.5%, 1.0%, 1.5%, and 2.0% respectively). The freezing storage time at -18℃ was set to 10 days, 20 days, and 30 days respectively, and all other aspects were the same as in Example 4. Then, the frozen dough from different groups was tested as follows: (1) Determination of rheological properties of frozen dough Rheological frequency scanning was employed. The dough was placed in the center of the test platform (1.3 mm gap), excess sample at the edges was trimmed, and the sample was sealed with mineral oil and equilibrated for 5 min. Frequency scanning from 0.1 to 10 Hz was performed at 25℃, and the dynamic changes of storage modulus (G') and loss modulus (G'') were recorded simultaneously. Rheological properties are one of the important indicators of dough functional properties, reflecting the viscoelasticity of the dough and having a significant impact on the processing quality and product quality of dough products. Storage modulus (G') reflects the elastic properties of dough; the higher the storage modulus, the better the elasticity of the dough. Loss modulus (G'') reflects the viscous properties of dough; the higher the loss modulus, the greater the viscosity of the dough.

[0082] The results are as follows Figure 15 It can be seen that adding wheat bran polysaccharides can change the rheological properties of dough. The storage modulus (G') and loss modulus (G'') of the polysaccharide dough are both greater than those of the control group (0.0%) wheat dough. This may be because polysaccharides have high water-holding capacity, which can lock in the moisture in the dough and prevent moisture loss. Under the same frozen storage time (10 d, 20 d, and 30 d), with the increase of wheat bran polysaccharide addition (0.5%~2.0%), the storage modulus (G') and loss modulus (G'') generally showed an upward trend, and the elasticity and viscosity properties of the dough were enhanced. The storage modulus (G') increased ( Figure 15 The results (A-10 d, A-20 d, A-30 d) indicate that the addition of wheat bran polysaccharides enhances the elasticity of the dough. This may be due to the interaction between polysaccharide molecules and proteins and other components in the dough, forming a more stable network structure; or it may be due to the dynamic filling effect of starch granules acting as a physical reinforcing phase to improve the mechanical strength of the gluten matrix, giving the dough higher resistance to deformation and elasticity. The loss modulus (G'') increased ( Figure 15 The results (B-10 d, B-20 d, B-30 d) indicate that the addition of wheat bran polysaccharides increased dough viscosity, possibly due to the increased internal friction and energy dissipation caused by the polysaccharide molecules. When the wheat bran polysaccharide addition was 1.5%–2.0%, the storage modulus (G') and loss modulus (G'') were significantly higher at 30 d (…). Figure 15 The levels remained high even after 30 days (A-30 d and B-30 d), indicating that the addition of wheat bran polysaccharides helps maintain the viscoelasticity of the dough after long-term frozen storage. With increasing wheat bran polysaccharide addition, the starch granule filling effect enhances the dough's binding force, promotes gluten matrix densification, and helps improve the dough's processing performance and product quality. These results demonstrate that the addition of wheat bran polysaccharides helps maintain the stability of the dough's rheological properties during frozen storage, providing a useful reference for the practical application of frozen dough.

[0083] (2) Laser confocal microscopy (CLSM) test of frozen dough Sample Preparation: Remove the sample and cut a 0.5×0.5×0.5 cm block from the center. Place the block on the cryostat holder and add a certain amount of tissue freezing medium. Place the block on the semiconductor freezing rack in the cryostat for 20 min. The temperature of the freezer and sample head should be -25℃. After freezing, fix the sample holder to the sample head. Trim the block and cut the sample into sections with a thickness of 50 μm. Attach the sections to a glass slide. Staining: Add 30 µL of a mixed staining solution (FITC, Rhodamine B, and Carcoflur fluorescent whitening agent (CW) working solutions with concentrations of 0.02%, 0.1%, and 0.01%, respectively) to the sections. Stain at room temperature in the dark for 10 min. Slowly add 100 µL of deionized water from one side, and blot the other side with absorbent paper. Observation: Cover with a coverslip and photograph using a laser confocal microscope. The excitation wavelengths for FTIC, Rhodamine B, and CW were 488 nm, 559 nm, and 405 nm, respectively, with corresponding emission wavelengths of 516 nm, 580 nm, and 432 nm. The magnification was 200 / 400x, and the scale bar was 100 µm.

[0084] Laser confocal microscopy (CLSM) utilizes laser excitation and scanning of fluorescent substances to non-invasively and at high resolution reveal the three-dimensional gluten network structure within dough through fluorescence reflectance imaging technology, providing a direct visual representation of the microscopic distribution and arrangement of gluten proteins. Fresh dough was used as the control group (FD, where fresh dough refers to dough that has not undergone freezing; the preparation method was the same as in Example 4, except that freezing was not performed and no wheat bran polysaccharides were added). Results are as follows... Figure 16 As shown, wheat bran polysaccharides have an improving effect on the gluten network structure of frozen dough. In fresh dough ( Figure 16 In the frozen dough images, a complete, continuous, and uniformly distributed gluten network structure can be clearly seen. With prolonged frozen storage time (10 d, 20 d, 30 d), the gluten network structure of the dough was gradually damaged. In the control group of frozen dough (0.0%), the gluten network showed obvious breaks and discontinuities, possibly due to the damaging effect of ice crystal formation and recrystallization on gluten proteins during freezing. When the amount of wheat bran polysaccharide added was 0.5%–1.5%, the continuity and integrity of the gluten network were improved. These results indicate that wheat bran polysaccharide can effectively stabilize the gluten network structure, reduce the damage of ice crystals to gluten proteins during freezing, and thus maintain the elasticity and extensibility of the dough. While adding 2.0% wheat bran polysaccharide showed some improvement, excessive amounts increased dough viscosity, affecting the uniformity of the gluten network and the processing characteristics of the dough. Therefore, adding an appropriate amount of wheat bran polysaccharide (0.5%–1.5%) can improve the gluten network structure of frozen dough and enhance its stability and freeze resistance.

[0085] (3) Determination of the freezeable water content of frozen dough The freezeable water content of dough was determined using differential scanning calorimetry (DSC). A quantitative amount of frozen dough was encapsulated in a DSC aluminum crucible. Using a blank crucible as a reference, the dough was brought to thermal equilibrium at -20°C for 2 minutes, followed by a programmed temperature increase (1°C / min) to 20°C, and the heat flow curve was recorded. The enthalpy of ice crystal melting was calculated using TA Universal Analysis software. The freezeable water content F in the frozen dough was determined. w The calculation formula is as follows: In the formula: H w The enthalpy of melting of the dough, J; For dough mass, g; W A The moisture content of the dough is expressed in g / g.

[0086] The freezeable water content in dough is an important indicator of frozen dough quality. During frozen storage, the freezeable water content directly affects the formation, growth, and recrystallization of ice crystals, thereby disrupting the integrity of the gluten protein structure. As storage time increases, ice crystals gradually accumulate in the dough, reducing the binding of protein and water molecules, causing more ice crystals to precipitate and grow, thus damaging the gluten network structure.

[0087] The results are as follows Figure 17 As shown, with the extension of freezing time (10 d, 20 d, 30 d), the content of freezeable water in the frozen dough gradually increased, but the increase rate was slower in the frozen dough after adding wheat bran polysaccharide compared to the control group. This may be because wheat bran polysaccharide can slow down the growth of ice crystals, resulting in smaller ice crystals, and the higher the amount of polysaccharide added, the more significant the inhibitory effect. P <0.05). During freezing for 10, 20, and 30 days, different proportions of polysaccharide addition significantly reduced the freezeable water content in the frozen dough. P <0.05%), at 2.0%, the freezeable water content decreased by 10.2%, 9.9%, and 14.4%, respectively. This may be because the polysaccharide can bind tightly to water molecules, forming a protective film that inhibits the formation of ice crystals in the dough. While adding wheat bran polysaccharide cannot completely prevent the increase in freezeable water content, it significantly reduces its growth trend. P <0.05), indicating that wheat bran polysaccharides can enhance the water-binding strength in frozen dough, increasing the dough's water-holding capacity and storage stability.

[0088] (4) Determination of the water flowability of frozen dough Low-field nuclear magnetic resonance (NMR) was used to analyze the moisture distribution of dough. A 1.6 g dough sample was weighed, encapsulated in a PTFE film, placed in an NMR tube, and positioned at the center of the RF coil for CPMG (Carr-Purcell-Meiboom-Gill Sequence) detection. Instrument parameters were set as follows: sequence mode CPMG; repetition time (TR) 2000 ms; echo time (TE) 0.100 ms; echo number 3500; spectral width 200 kHz; gain 3; accumulation count 32.

[0089] The moisture content and distribution of dough have a crucial impact on maintaining the quality of frozen dough products. The shorter the relaxation time, the more tightly the moisture in the dough is bound to the gluten protein or starch, and the free sulfhydryl groups effectively restrict the flow and migration of moisture.

[0090] The results are as follows Figure 18 As shown, regardless of the addition of wheat bran polysaccharide, the signal amplitude gradually increased with prolonged frozen storage time (10 d, 20 d, 30 d). When the wheat bran polysaccharide addition was 0.5%, the signal amplitude of the frozen dough (10 d) was higher than that of the control group, but the signal amplitude of the frozen dough (20 d, 30 d) was lower than that of the control group. This indicates that adding 0.5% wheat bran polysaccharide can reduce the signal amplitude of the dough during frozen storage, and the water flowability of the dough is improved. This may be because wheat bran polysaccharide can effectively stabilize the gluten network structure and reduce the damage of ice crystals to the gluten network structure, thereby reducing water flowability. To improve the water flowability of frozen dough and enhance its stability and quality, the recommended addition amount of wheat bran polysaccharide is about 0.5%.

[0091] (5) Determination of dough farinograph properties Wheat bran polysaccharide was mixed evenly with wheat flour to prepare mixed powders with wheat bran polysaccharide addition amounts of 0.0%, 0.5%, 1.0%, 1.5% and 2.0%, respectively. The powder quality characteristics were measured using a Mixolab mixing tester.

[0092] Increased water absorption can increase the yield of flour products. Dough formation time has a certain impact on dough elasticity. Wheat flour with high gluten content and excellent quality has stronger gluten, resulting in a longer dough formation time, and vice versa. The longer the dough's stability time, the better its toughness, the greater the gluten strength, and the better its performance in the dough processing process. Protein weakening degree reflects the dough's ability to withstand mechanical mixing; the lower the protein weakening degree, the stronger the gluten, and the better the dough's workability.

[0093] The results are shown in Table 7. With the sequential addition of 0.0% to 2.0% wheat bran polysaccharide, the water absorption rate of the wheat dough increased from 56.40% to 58.60%. The dough formation time and stabilization time showed a trend of first increasing and then decreasing. The protein weakening degree was significantly reduced. The differences between different amounts of wheat flour added were statistically significant. P <0.05). The increased water absorption rate may be due to the increased water-holding capacity of gluten protein; it may also be due to the strong hydrogen bond interactions of wheat bran polysaccharides, which can generate more hydrophilic surfaces. The strong water absorption capacity of polysaccharides promotes the increase of dough water absorption rate, slows down starch retrogradation in flour products, and can extend the shelf life of products. The dough formation time and stability time initially increase may be because wheat bran polysaccharides contain disulfide bonds, which can promote the aggregation of gluten protein, thereby strengthening the gluten network structure. However, when the addition amount is too high, the gluten protein is diluted. At this time, the dilution effect is greater than the disulfide bond effect, so the formation time and stability time show a decreasing trend.

[0094] When the amount of wheat bran polysaccharide added was 1.0%, the dough formation time and stability time reached their maximum values ​​of 3.16 min and 7.25 min, respectively. The increased dough formation and stability times after adding an appropriate amount of wheat bran polysaccharide may be due to the competition for water molecules among polysaccharides, starch granules, and gluten proteins, prolonging protein hydration and starch swelling, thus increasing the dough formation time. Alternatively, it may be because the polysaccharide is rich in hydroxyl groups, which enhances hydrogen bonding interactions in the dough, increasing the strength of the gluten structure and thus prolonging the dough stability time. These results indicate that appropriate addition of wheat bran polysaccharide can improve dough stability and enhance its processing properties.

[0095] Table 7 Effects of wheat bran polysaccharides on dough farinograph properties

[0096] Note: Different lowercase letters in the same column indicate significant differences between data. P <0.05) (6) Extraction and freeze-thaw treatment of gluten protein Weigh 500 g of wheat flour and mix it with 30°C warm water using a dough mixer. The shaped dough is then placed in a 30°C / 85% (RH) constant temperature and humidity chamber for 20 minutes to complete gluten hydration. The dough is then repeatedly kneaded and washed with distilled water (until the washing liquid becomes clear). The wet gluten is then quick-frozen at -80°C, freeze-dried, pulverized through a 100-mesh sieve, and stored at 4°C for later use.

[0097] Wheat bran polysaccharide and gluten protein powder were mixed evenly to prepare mixed powders of 0.0%, 0.5%, 1.0%, 1.5%, and 2.0% respectively. 5.0 g of the mixed powder was magnetically stirred with 20 mL of distilled water for 1 h, then hydrated at 30℃ / 85% RH for 16 h. After centrifugation at 4000 r / min for 20 min, samples were processed as follows: 1) A portion of the wet gluten was directly freeze-dried and pulverized (100 mesh) for water retention testing; 2) The remaining samples underwent 5 freeze-thaw cycles (rapid freezing at -40℃ for 1 h, then storage at -18℃ for 23 h, followed by thawing at 30℃ / 85% RH for 1 h per cycle) before freeze-drying. All samples were sieved through a 100-mesh sieve and stored at 4℃.

[0098] Gluten protein water-holding capacity test: Add 50.00 mg of gluten protein complex powder (m2) to a centrifuge tube (m1), then add 4.00 mL of distilled water and vortex to disperse thoroughly. After standing for 1 h of hydration, centrifuge at 5000 r / min for 10 min to remove the supernatant, and weigh the mass of the hydrated gluten protein complex powder (M). The calculation formula is as follows: Gluten protein water-holding capacity (g / g) In the formula: m1 is the mass of the centrifuge tube, g; m2 is the mass of the gluten protein composite powder, g; M is the mass of the gluten protein composite powder hydrate, g.

[0099] Water-holding capacity is an important functional property of gluten proteins, reflecting to some extent the uniformity and stability of the gluten network structure. It is a crucial factor in assessing the water interaction within the polysaccharide, gluten, and dough system. The water-holding capacity of dough significantly impacts the texture, mouthfeel, and overall quality of the product. Results are as follows... Figure 19 As shown, with the increase of wheat bran polysaccharide addition, the water-holding capacity of the gluten protein system significantly increased ( P <0.05). This may be due to the abundance of hydroxyl structures in wheat bran polysaccharides, which have strong water absorption and retention properties; it may also be due to the complex structure formed by the interactions between polysaccharide molecules, water molecules, and gluten protein molecules through hydrogen bonds, which can bind some water molecules, making the gluten protein skeleton structure more stable, thereby enhancing its water-holding capacity.

[0100] Determination of free sulfhydryl content in gluten protein: Transfer 5.00 mL of Tris-glycine buffer (10.40 g Tris, 480.00 g urea, 10.40 g glycine, and 1% sodium dodecyl sulfate (SDS) solution to a centrifuge tube, add 150.00 mg of the composite powder and vortex mix. Add 0.1 mL of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) colorimetric reagent (40 mg DTNB dissolved in 1% SDS solution and diluted to 10 mL), centrifuge at 4500 r / min for 20 min, and measure the absorbance of the supernatant at 412 nm, using deionized water as a blank reference.

[0101] In the formula: A 412 λ is the absorbance at 412 nm; C is the sample concentration (mg / mL); 5.02 is the dilution factor.

[0102] Free thiol groups play a crucial role in the formation of gluten networks. They are precursors to disulfide bonds and promote the cross-linking and aggregation of gluten proteins through thiol oxidation and disulfide bond exchange reactions, thereby enhancing the strength and stability of the gluten network structure. This is conducive to the formation of a stable and continuous gluten network structure. An appropriate amount of free thiol content helps improve the elasticity and toughness of flour products and affects the processing quality of flour products. Therefore, the free thiol content is often used to reflect the dynamic changes of disulfide bonds.

[0103] The results are as follows Figure 20As shown, the changes in free sulfhydryl content in gluten protein under freeze-thaw and freeze-thaw cycle treatments exhibited similar trends, initially decreasing and then increasing. When the amount of wheat bran polysaccharide added was 0.5%–1.0%, the free sulfhydryl content decreased. Intermolecular disulfide cross-linking causes gluten protein to form large molecular weight polymers. When the addition amount was 1.0%–2.0%, the free sulfhydryl content increased. This may be because excessively high free sulfhydryl content leads to physical entanglement between polysaccharide molecules and gluten protein macropolymers, interfering with gluten structure and hindering the formation of a gluten network. This is not conducive to forming a tight and orderly gluten network structure, indicating that appropriate addition of wheat bran polysaccharide can effectively reduce disulfide bond breakage, making the gluten protein structure more stable. After freeze-thaw cycle treatment, the increase in free sulfhydryl content significantly decreased. This may be because high wheat bran polysaccharide content can inhibit ice crystal recrystallization, weakening the damage of ice crystals to the gluten protein network structure during freeze-thaw processes and delaying the depolymerization process between gluten protein molecules. Compared to unfrozen gluten protein, freeze-thaw treatment resulted in an overall decrease in the free sulfhydryl content of gluten protein. This is likely because the formation and melting of ice crystals during the freeze-thaw process damaged the gluten protein structure, exposing and oxidizing some sulfhydryl groups, thus reducing the free sulfhydryl content. With increasing amounts of wheat bran polysaccharide, the difference in free sulfhydryl content between freeze-thawed and unfrozen gluten protein gradually decreased. This is likely because wheat bran polysaccharide, to some extent, protected the gluten protein structure and reduced the loss of sulfhydryl groups during the freeze-thaw process.

[0104] Determination of the secondary structure of gluten protein: Accurately weigh 2.00 mg of gluten protein sample and 200 mg of potassium bromide powder, grind and homogenize under an infrared lamp, and then press into transparent thin sheets. Use a Fourier transform infrared spectrometer to analyze the samples from 4000 to 400 cm⁻¹. -1 Wavenumber range spectral acquisition (resolution 4.0 cm⁻¹) -1 (32 scans). After baseline correction and Gaussian smoothing preprocessing using OMNIC software, the amide I band was deconvolved. Combined with the second derivative peak fitting technique of Systat PeakFit software, the content of each secondary structure in the gluten protein sample was analyzed.

[0105] The spatial conformation of proteins plays a decisive role in their physiological activity and functional properties. α - spiral and β - Folds are the most stable structures in proteins, among which α - The helical spatial conformation helps enhance the overall elasticity of the dough. The results are shown in Table 8. Regardless of whether there were 0 freeze-thaw cycles or 5 freeze-thaw cycles, with the increase of wheat bran polysaccharide addition... α - spiral and β - The folding content showed an increasing trend. β- The content of both corner and irregular curls showed a decreasing trend. This may be because the hydroxyl groups in wheat bran polysaccharides form strong hydrogen bonds with gluten protein molecules, affecting the hydration level of gluten protein, leading to partial dehydration and changes in its secondary structure. Adding wheat bran polysaccharides can slow down the dehydration of gluten protein. α - spiral and β The reduction in folded structure may be due to the interaction between wheat bran polysaccharide and gluten protein, which can inhibit ice crystal growth and form a protective layer, thereby reducing the damage to the gluten protein structure during freeze-thaw and enhancing the freeze-thaw stability of gluten.

[0106] Table 8. Effects of wheat bran polysaccharides on the secondary structure of gluten protein.

[0107] Note: Different lowercase letters in the same column indicate significant differences between data. P <0.05) The above results indicate that the addition of wheat bran polysaccharides increased the storage modulus (G') and loss modulus (G'') of the frozen dough, enhanced its viscoelasticity, and significantly increased its water absorption. P <0.05, the weakening degree decreased significantly ( P <0.05%, dough moisture fluidity increased, and the water-holding capacity of the gluten protein system was significantly improved ( P <0.05), α - spiral and β - The content of folded structures is increasing. β - The content of corners and irregular curls decreased; when the addition amount was 1.0%, the dough formation time increased from 2.76 min to 3.16 min, and the stability time increased from 6.40 min to 7.25 min.

[0108] Example 6 A frozen dough steamed bun, prepared as follows: The frozen dough from Example 4 was refrigerated at 4°C for 4 hours and then thawed at room temperature for 40 minutes. It was then transferred to a constant temperature and humidity proofing box (37°C / 85% RH) for 1 hour of proofing. It was then steamed in a 2200 W induction cooker for 25 minutes and then simmered for 5 minutes.

[0109] Experimental Example 6 The difference from Example 6 is that the amount of wheat bran polysaccharide added to the frozen dough in Example 1 was 0.0g, 0.5g, 1.0g, 1.5g, and 2.0g (calculated based on the amount of wheat flour added, the proportions of wheat bran polysaccharide added were 0.0%, 0.5%, 1.0%, 1.5%, and 2.0%, respectively), and the freezing storage time at -18℃ was set to 10 days, 20 days, and 30 days, respectively. All other aspects were the same as in Example 6. After preparing frozen dough steamed buns with different amounts of wheat bran polysaccharide and different freezing storage times, the steamed buns were covered with gauze to prevent moisture evaporation and kept at room temperature for 1 hour until the steamed buns were completely cooled. The following measurements were then taken (fresh steamed buns (CK) were used as the control group; fresh steamed buns refer to steamed buns made from fresh dough (without added wheat bran polysaccharide)): (1) Determination of specific volume and height-to-diameter ratio of steamed buns After the steamed buns have cooled to room temperature, weigh them and measure their mass M (g). Measure their volume V (mL) using the volume substitution method.

[0110] The height and diameter of the steamed bun were measured using vernier calipers, and the height-to-diameter ratio was calculated as shown below.

[0111] Specific volume, as one of the most important sensory characteristics of steamed buns, can directly reflect the quality characteristics of steamed buns and is a comprehensive manifestation of the changes in frozen dough steamed buns. The results are shown in Table 9. Compared with fresh steamed buns (CK), the specific volume and height-to-diameter ratio of frozen dough steamed buns were significantly reduced. P <0.05), but with the increase of wheat bran polysaccharide addition, the specific volume and aspect ratio of the steamed buns improved. With the increase of wheat bran polysaccharide addition, the specific volume and aspect ratio of the steamed buns showed a trend of first increasing and then decreasing. When the wheat bran polysaccharide addition was 1.0%, the specific volume and aspect ratio of the steamed buns were optimal, at 2.72 mL / g and 0.71%, 2.56 mL / g and 0.70%, and 2.39 mL / g and 0.63%, respectively. This may be because wheat bran polysaccharide can enhance the gluten protein network structure of the dough, improve the extensibility and gas retention of the dough, and is beneficial to the expansion and shaping during dough fermentation. The results of dough rheological properties also showed that excessive wheat bran polysaccharide addition would reduce the viscoelasticity of the dough and inhibit fermentation expansion, resulting in a smaller volume and increased chewiness of the steamed buns. As shown in Table 9, during the storage period of 10-30 days, the specific volume of the frozen dough with added wheat bran polysaccharide was consistently significantly higher than that of the control group with 0.0% addition (…). P <0.05), indicating that although this polysaccharide cannot completely prevent the deterioration of frozen dough quality, it can effectively slow down the deterioration rate, improve the storage stability of frozen dough, and improve the quality of steamed buns.

[0112] Table 9. Effects of wheat bran polysaccharide on specific volume and height-to-diameter ratio of frozen dough steamed buns.

[0113] Note: Different lowercase letters in the same column indicate significant differences between data. P <0.05) (2) Determination of the color of steamed buns The color difference was measured using a Konica Minolta CR-410 colorimeter, based on the CIE-Lab color space representation. The value represents brightness, a The value reflects the red-green balance, b The value indicates the degree of yellow-blue. Each sample was measured three times and the average value was taken.

[0114] Color is an important indicator affecting the sensory appeal of steamed buns. During frozen storage, enzymatic browning by polyphenol oxidase and Maillard reactions involving amino carbonyl groups in the dough lead to a decrease in the color of the steamed bun slices. Moisture loss during frozen storage also reduces the gloss of the dough. The results are as follows... Figure 21 As shown, with increasing freezing time, compared with the control group with 0.0% added wheat bran polysaccharide, the L of the steamed buns after adding wheat bran polysaccharide increased. value and a value( Figure 21 A and B in the middle are significantly reduced, b value( Figure 21 C in the middle is significantly increased ( P <0.05). This indicates an increase in the yellowness of the steamed buns. This may be due to the fact that the wheat bran polysaccharide raw material itself is pale yellow, and its addition reduces the whiteness of the flour. It may also be due to the gelatinization reaction of the polysaccharides during the steaming process, forming caramel compounds and deepening the color of the steamed buns. With the extension of frozen storage time (10 days, 20 days, 30 days), the L of the steamed buns... value, a value and b The values ​​all showed a decreasing trend, indicating that frozen storage would affect the color of the steamed buns.

[0115] (3) Determination of the texture of steamed buns The textural properties of steamed buns were determined using a texture analyzer. Cooled steamed buns were prepared into 20 mm thick standard samples, and a P32 cylindrical probe was used for total texture analysis (TPA). Parameter settings: pre-compression speed 2.00 mm / s, testing speed 5.00 mm / s, reset speed 5.00 mm / s; deformation 50%, trigger threshold 5 g, interval between two compressions 5 s. Hardness, elasticity, resilience, and chewiness parameters were simultaneously measured through a single compression cycle.

[0116] Within a certain range, the textural properties of steamed buns can reflect their quality characteristics and demonstrate the storage stability of frozen dough. Hardness and chewiness are important indicators for evaluating flour products. The lower the hardness and chewiness, the softer the steamed bun and the better its taste. The elasticity of dough is closely related to fermentation capacity, yeast activity, and carbon dioxide release. Elasticity refers to the ability of steamed buns to return to their original state after compression, while resilience is the ability to recover after compression.

[0117] The results are as follows Figure 22 As shown, during frozen storage, with the extension of freezing time (10 days, 20 days, 30 days), when the amount of wheat bran polysaccharide added was 0.5%~2.0%, the hardness and chewiness of the steamed buns showed an increasing trend, while the elasticity and resilience showed a decreasing trend. When the amount of wheat bran polysaccharide added was 0.5%~1.0%, the elasticity and resilience of the frozen dough steamed buns were significantly higher than those of the control group with an addition of 0.0% (…). P The concentration of wheat bran polysaccharide (<0.05) indicates that adding an appropriate amount of wheat bran polysaccharide can delay the deterioration of frozen dough steamed buns. This may be because wheat bran polysaccharide has good hydrophilicity and stability, which can inhibit water loss and ice crystal formation during freezing, thereby protecting the gluten network structure. It may also be because wheat bran polysaccharide contains dietary fiber, and the porous structure of dietary fiber can make the dough highly absorbent. It can interact with components such as gluten protein, starch and water, which can enhance the density and elasticity of the gluten protein network structure, improve the water retention capacity of the steamed buns, and improve the textural properties of the steamed buns.

[0118] (4) Sensory evaluation of steamed buns The sensory evaluation was conducted according to the GB / T 35991-2018 sensory evaluation table. Twenty professionally trained personnel were selected to evaluate the specific volume, appearance, structure, elasticity, and taste of the steamed buns. The evaluation criteria are shown in Table 10.

[0119] Table 10 Scoring Table for Wheat Bran Polysaccharide Steamed Buns

[0120] The sensory quality of steamed buns was evaluated using sensory descriptive analysis, and the results are as follows: Figure 23As shown in the figure, the total sensory evaluation score of fresh steamed buns reached 85.80 points, higher than that of frozen steamed buns. After freezing, the elasticity, surface gloss, structural integrity (surface and interior), taste, and overall score of the steamed buns all showed a downward trend. During frozen storage, the sensory scores of steamed buns with added wheat bran polysaccharides were consistently better than those of the control group (0.0%) without added wheat bran polysaccharides. Furthermore, when the addition amount was 1.0%, the total sensory evaluation score was the highest, reaching 84.00, 85.20, and 85.35 points at the three time points, respectively. Rheological properties, microstructure observation, specific volume, and texture determination experiments further confirmed that the frozen dough of the control group (0.0%) without added wheat bran polysaccharides was prone to problems such as gluten network collapse, yeast activity inhibition, and moisture loss, leading to a significant deterioration in steamed bun quality. Based on the above results, the optimal addition amount of wheat bran polysaccharides is 1.0%, which can effectively alleviate freezing damage and maintain the sensory quality and overall quality of the steamed buns.

[0121] (5) Determination of the microstructure of steamed buns After freeze-drying, the steamed buns were sprayed with gold, and their internal microstructure was observed under a scanning electron microscope (SEM).

[0122] The results are as follows Figure 24 As shown, the gluten network structure of steamed buns changed significantly with increasing wheat bran polysaccharide addition. Under the same freezing time, the gluten network structure of the control group (0.0%) was relatively loose, with larger and unevenly distributed pores. With increasing wheat bran polysaccharide addition (0.5%~2.0%), the gluten network structure gradually became denser, with smaller and more uniform pores, resulting in a more complete gluten network structure. This indicates that the addition of wheat bran polysaccharide can improve the gluten network structure of steamed buns, making it denser and more stable, thus enhancing the freezing resistance of the steamed buns and mitigating the destructive effect of freezing time on the gluten network structure. Under the same freezing time (10 d, 20 d, 30 d), the degree of damage to the gluten network structure gradually decreased with increasing wheat bran polysaccharide addition. With prolonged freezing time, under the same amount of wheat bran polysaccharide added, the gluten network structure of fresh steamed buns (FSB) remained intact and continuous, with smaller pores. After 10 days of frozen storage, the gluten network structure began to show some damage, with larger pores. After 20 days of frozen storage, the damage to the gluten network structure intensified, and the pores became more irregular. After 30 days of frozen storage, the gluten network structure became significantly looser, with larger and unevenly distributed pores, indicating that freezing time has a certain destructive effect on the gluten network structure of steamed buns.

[0123] (6) X-ray diffraction (XRD) analysis Frozen dough steamed buns were freeze-dried in a vacuum freeze dryer, and the samples were pulverized and sieved (100 mesh) for later use. XRD analysis was performed using a D8 ADVANCE diffractometer (copper target, nickel filter, 27 mA, 50 kV) with scanning parameters of 5–50° (2θ), a step size of 0.02°, and a rate of 6° / min. XRD patterns were analyzed using Jade 6.0 software.

[0124] X-ray diffraction is widely used to study the crystal properties of starch granules. The height and width of the diffraction peaks depend on the content and size of the starch crystals. The higher the crystal content and the greater the integrity of the crystalline regions in the system, the narrower and higher the diffraction peaks will be. Results are as follows... Figure 25 As shown, the steamed bun exhibits characteristic diffraction peaks near 2θ = 20°, 19°, and 18°, which coincide with the characteristic peaks of starch type A crystals. Most grains (such as wheat and rice) typically exhibit the characteristic crystal form of type A. With increasing wheat bran polysaccharide addition, the crystallinity of the frozen polysaccharide steamed bun was lower than that of the control group (0.0%) without added wheat bran polysaccharide. This may be because the addition of wheat bran polysaccharide increases the proportion of amorphous substances in the steamed bun. Steamed buns are mainly composed of amorphous starch and other carbohydrates, and the internal atomic or molecular arrangement of amorphous substances is relatively disordered, causing the peaks to become more diffuse and the crystallinity to decrease accordingly. Alternatively, it may be due to the polyhydroxy structure of wheat bran polysaccharide, which can bind to the exposed carbonyl groups at the ends of starch molecular chains. This binding effectively inhibits the interaction between starch molecular chains, hindering the recrystallization process and delaying the formation of the double helix structure.

[0125] The above results indicate that with increasing wheat bran polysaccharide addition, the yellowness value of steamed buns increased, while the redness value decreased. The hardness, elasticity, resilience, and chewing texture of frozen dough steamed buns were all improved. At an addition level of 1.0%, the specific volume of steamed buns stored frozen for 10, 20, and 30 days increased from 2.50 mL / g to 2.72 mL / g, 2.26 mL / g to 2.56 mL / g, and 1.65 mL / g to 2.39 mL / g, respectively. The aspect ratio increased from 0.67% to 0.71%, 0.65% to 0.70%, and 0.59% to 0.63%, respectively. Sensory scores increased from 64.50 to 84.00, 60.80 to 85.20, and 69.35 to 85.35, respectively. This suggests that wheat bran polysaccharide can be used as a novel antifreeze agent for frozen dough, and the optimal addition level is 1.0%. In addition, although wheat bran polysaccharides can improve the storage stability of frozen dough, excessive addition can have an adverse effect on the dough structure, thereby reducing the quality of steamed buns.

[0126] Comparative Example 1 The difference from Example 1 is that ultrasonic extraction was not performed using an ultrasonic processor, but rather enzymatic hydrolysis with cellulase at 50°C for 21 minutes. All other aspects are the same as in Example 1.

[0127] Comparative Example 2 The difference from Example 1 is that no cellulase was added; otherwise, it is the same as Example 1.

[0128] Experimental Example 7 The extraction rates of wheat bran polysaccharides in Example 1, Comparative Example 1, and Comparative Example 2 were determined using the same method as in Example 1 (1). Three parallel experiments were conducted for each group simultaneously, and the final results were averaged. The results are shown in Table 11. This indicates that the present invention utilizes ultrasound and enzymes for synergistic extraction to prepare wheat bran polysaccharides, achieving a significant synergistic effect.

[0129] Table 11 Extraction rates of wheat bran polysaccharides from different groups

[0130] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing wheat bran polysaccharides that can improve the storage stability of frozen dough and the quality of frozen dough products, characterized in that, The process includes the following steps: mixing defatted wheat bran with water and cellulase, extracting by ultrasonication, inactivating the enzyme, taking the supernatant, and drying to obtain wheat bran polysaccharide; the mass-volume ratio of defatted wheat bran to water is 1 g: 15 mL~35 mL; the addition ratio of defatted wheat bran to cellulase is 1 mg: 4500 U~5500 U.

2. The preparation method according to claim 1, characterized in that, The ultrasonic extraction time is 10 min to 30 min, the ultrasonic extraction temperature is 40℃ to 60℃, and the ultrasonic intensity is 300 W to 600 W.

3. The preparation method according to claim 1, characterized in that, The preparation method of defatted wheat bran includes the following steps: crushing and sieving wheat bran to obtain wheat bran powder; mixing and stirring the wheat bran powder with n-hexane, collecting the precipitate, and obtaining defatted wheat bran.

4. The preparation method according to claim 3, characterized in that, The sieving process is a 50-70 mesh sieve; the mass-to-volume ratio of wheat bran powder to n-hexane is 1 g: 2 mL-5 mL; the wheat bran is the wheat bran of Yongliang No. 4 wheat from the Hetao region.

5. A wheat bran polysaccharide that can improve the storage stability of frozen dough and the quality of frozen dough products, characterized in that, It is prepared by the preparation method described in any one of claims 1 to 4.

6. The preparation method according to any one of claims 1 to 4 or the application of the wheat bran polysaccharide according to claim 5 in the preparation of an antifreeze agent for frozen dough.

7. The preparation method according to any one of claims 1 to 4 or the application of the wheat bran polysaccharide according to claim 5 in the preparation of frozen dough.

8. The application according to claim 7, characterized in that, The mass ratio of wheat bran polysaccharide to wheat flour is 0.5~1.5:100; the wheat bran polysaccharide can improve the storage stability of frozen dough; Improving storage stability includes: reducing the freezeable water content of frozen dough, increasing water flowability, and enhancing the water-holding capacity of gluten proteins; enhancing the viscoelasticity of frozen dough, improving the continuity and integrity of the gluten protein network structure, and increasing the water absorption rate of frozen dough; extending the formation time and stabilization time of frozen dough; reducing the content of free thiol groups, increasing the content of disulfide bonds, and increasing the content of α-helical and β-sheet structures in gluten proteins.

9. The preparation method according to any one of claims 1 to 4 or the application of the wheat bran polysaccharide according to claim 5 in the preparation of frozen dough steamed buns.

10. The application according to claim 9, characterized in that, The mass ratio of wheat bran polysaccharide to wheat flour is 0.5~1.5:100; the wheat bran polysaccharide can improve the hardness, elasticity, resilience and chewiness of frozen dough steamed buns, and can increase the yellowness value and decrease the redness value of steamed buns.

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